PROJECT 5 ? PROJECT SUMMARY/ABSTRACT Epstein-Barr virus (EBV) latent infection of B lymphocytes in vitro results in their growth transformation; however, in vivo, the growth of EBV-infected lymphocytes is normally constrained by robust immune responses against viral antigens. As a result, EBV genes essential for growth in vitro (such as LMP1 and EBNA2) are often not expressed in EBV-positive lymphomas such as Burkitt lymphoma (BL), Hodgkin Disease and diffuse large B cell lymphomas. Thus, in vitro transformation studies cannot adequately model how EBV infection promotes common types of EBV-positive human lymphomas that have more stringent forms of viral latency. The EBV BARTs microRNAs, and EBNA3A, are amongst the very few EBV-encoded genes/microRNAs expressed in human BLs, and are likely to play important ?driver? roles in this type of lymphoma. We have shown that BARTs play an important role in maintaining the viability of EBV-positive BLs in vitro, and in decreasing the immunogenicity of EBV-transformed lymphoblastoid cell lines. The latent EBNA3A protein encodes a transcription factor that is essential for in vitro growth of EBV-transformed B cells, and is thought to collaborate with the closely related EBNA3C protein to inhibit expression of important tumor suppressors (including p16, p15 and BIM) by inducing EZH2-mediated H3K27 trimethylation of their promoters. However, the roles of BARTs and EBNA3A in promoting EBV-induced lymphomas in vivo have not been well studied, particularly in the context of lymphomas with more stringent latency. EBV-infected humanized mice provide sophisticated models for understanding the complex interactions between EBV, T cells, cellular pathway alterations and the microenvironment. We have recently developed a new cord blood- humanized mouse model that allows EBV mutants that are non-transforming in vitro (including EBNA2- deleted EBV) to form lymphomas with stringent viral latency in vivo. We propose to use two different humanized mouse models to examine the roles of BARTs and EBNA3A for EBV-induced lymphomas in vivo, and to determine if drugs which block essential EBNA3A functions inhibit lymphoma development. In Aim 1, we will examine how loss of BARTs affects viral pathogenesis in the context of type III versus Wp-restricted viral latency, and examine potential mechanisms by which BARTs expression is upregulated in vivo. In Aim 2, we will use the cord blood-humanized mouse model to explore the role of the EBNA3A protein in vivo. In Aim 3, we will explore the therapeutic potential of drugs (CK4/6 and EZH2 inhibitors) that block essential EBNA3A/3C-regulated pathways. This project interacts extensively with Projects 3 and 4, and uses the cores for immunohistochemistry, bioinformatics, and generation of EBV mutant genomes. The results of these studies should provide key insights into the mechanism(s) by which stringent EBV infection causes lymphomas in vivo, and may identify new therapeutic approaches for treating EBV-induced lymphomas.